Separation and Purification Technology 118 (2013) 835–841

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Separation and Purification Technology

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Concentration and purification of a-galactosidase from watermelon

(Citrullus vulgaris) by three phase partitioning
Hasan Bayraktar, Seçil Önal ⇑
_
Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova-Izmir, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: Three-phase partitioning (TPP) was used to concentrate and purify a-galactosidase from watermelon
Received 3 April 2013 (Citrullus vulgaris). The various process parameters required for efficient purification of a-galactosidase
Received in revised form 12 August 2013 were optimized to get highest purity fold and yield. The best a-galactosidase yield (76.7%) with a nearly
Accepted 26 August 2013
2.7-fold purification was obtained in the interphase of the TPP system, which consisted of the crude
Available online 5 September 2013
extract to t-butanol ratio of 1:1 (v/v) in the presence of 50% (w/v) (NH4)2SO4 at pH 5.5. The sodium dode-
cyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis revealed a substantial level of puri-
Keywords:
fication of a-galactosidase from watermelon. The molecular weight of the enzyme was determined
Three-phase partitioning (TPP)
a-Galactosidase approximately as 45 kDa. The purified enzyme was characterized with respect to its activity and stability.
Watermelon Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and
Citrullus vulgaris stability were studied. Optimum pH and temperature of a-galactosidase were determined as pH 6.0 and
Purification 60 °C, respectively. The purified enzyme was also very stable at a temperature range of 4–50 °C and a pH
range of 4.0–6.5. The Km and Vmax values were calculated from Lineweaver–Burk plot as 0.14 mM and
0.12 U, respectively. The results indicated that, TPP is a simple, quick, economical and very attractive pro-
cess for primary purification of a-galactosidases compared to conventional chromatographic protocols.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Three phase partitioning (TPP) is a simple and often one-step

a-Galactosidase (a-D-galactoside galactohydrolase, EC 3.2.1.22)
is a member of hydrolase class that catalyzes the hydrolysis of ter-
minal a-1,6-linked D-galactose residues in galactooligosaccharides
and galactopolysaccharides [1–3]. a-Galactosidases are mainly de-
rived from plant, animal and microbial sources. They are important
industrial enzymes with numerous biotechnological applications
such as; in sugar, pulp and paper industries, enzymatic synthesis,
structural analysis, feed processing, blood group transformation
and treatment of Fabry’s disease [4–10]. There are several reports
about purification and characterization of a-galactosidases from
different enzyme sources employing various traditional purifica-
tion processes. These processes involve precipitation and chro-
matographic techniques such as; hydrophobic interaction, jel
filtration and affinity chromatography [11–14]. Most of these sep-
aration and purification techniques are generally involved a num-
ber of steps, scalling up is difficult and also expensive. Therefore,
three phase partitioning (TPP) was used an alternative method
for purification of a-galactosidase to solve mentioned drawbacks.
⇑ Corresponding author. Tel.: +90 232 343 86 24; fax: +90 232 311 54 85.
E-mail addresses: hbayraktar@ymail.com (H. Bayraktar), secil.onal@ege.edu.tr
(S. Önal).
procedure successfully used for separation and purification of en-
zymes and proteins in recent years. It involves the addition of a salt
to the aqueous solution containing proteins followed by the addi-
tion of a water miscible aliphatic alcohol. In less than an hour three
phases are formed. The upper solvent phase containing pigments,
lipids, hyrophobic materials is separated from lower aqueous
phase containing proteins, saccharides and cell debris by an inter-
mediate layer. This protein-rich middle layer generally contains
desired enzymes or proteins [15–18]. TPP process is quite complex
and involve ionic strength and exclusion-crawding effects, kosmo-
tropy, osmotic stress, cavity surface tension enhancement and con-
formational protein tightening. It is also simple, inexpensive,
scalable and rapid procedure that works at room temperature in
comparison to conventional separation and purification processes.
TPP can also be used directly with the crude suspensions. As re-
ported by several researchers, besides of the physical conditions
of the assay, the partitioning process is also affected by the hydro-
philicity, the moleculer weight and pI of protein [18–21].
TPP has been used to purify a number biomolecules with high
recovery and purity levels like; protease from papaya peels [22],
giant catfish [23] and Calotropis procera latex[24], invertase from
tomato [25], Baker’s yeast [26] and Aspergillus oryzae[19], laccase
from Ganoderma sp. WR-1[27], a-galactosidases from A. oryzae[19],
pepino [28] and tomato [29], a-amylase inhibitor from bean seeds

1383-5866/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.08.040
836 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841
[30], forskolin from Coleus forskohlii roots [31], amylase/protease
inhibitor from Eleusine coracana[32].
The present work reports the separation, concentration and
purification of a-galactosidase from watermelon (Citrullus vulgaris)
using a one-step TPP. According to our knowledge, there are no any
reports on a-galactosidase purification from watermelon using TPP
in scientific literature. All TPP experiments were carried out with
the crude a-galactosidase extracted from watermelon. As the
physiological conditions affect partitioning of proteins during
TPP, the process was optimized to get high purification fold and
yield. For this purpose, the effect of various system parameters
like; salt type and its saturation, organic solvent type, ratio of en-
zyme amount to organic solvent and pH to the partitioning of
the enzyme was investigated. Biochemical properties of an enzyme
are very important for evaluation of its potential use especially in
biotechnological applications. Therefore, the purified enzyme was
also characterized with respect to its activity and stability at vari-
ous temperature and pH ranges. Sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis (SDS–PAGE) analysis and
determination of kinetic parameters (Km and Vmax) were also
carried.
2. Materials and methods
2.1. Materials
tert-Butanol and ammonium sulfate were pure grade and were
procured from E. Merck (Darmstad, Germany). p-Nitrophenyl-a-D-
galactopyranoside (PNPG) and Coomassie Brilliant Blue R-250 were
purchased from Sigma Chem. Co. (St. Louis, MO, USA). Fresh ripe
watermelon (Citrullus vulgaris) was purchased from a local market,
Turkey. All other chemicals and reagents were of the highest avail-
able purity and used as purchased.
2.2. Methods
2.2.1. Extraction of a-galactosidase from watermelon
After removal of the peels and the seeds, fresh ripe watermelon
(2200 g) flesh was homogenized with 0.05 M sodium citrate buffer
(pH 6.0). The homogenate was filtered from two layers of cheese-
cloth and then centrifuged at 10,000 rpm for 15 min at 4 °C (Het-
tich Universal 30 RF). The obtained supernatant was subjected to
85% (w/v) ammonium sulfate precipitation and then allowed to
stand overnight at 4 °C with continuous stirring. The precipitate
was collected by centrifugation at 10,000 rpm for 30 min at 4 °C.
The pellet was dissolved in sodium citrate buffer (0.05 M, pH 6.0)
and then dialyzed against the same buffer at 4 °C for overnight.
The dialysate represented as ‘‘crude watermelon a-galactosidase
extract’’ and used for further three-phase partitioning studies.
The protein content, activity and specific activity of the crude en-
zyme extract were determined as 1.34 mg/ml, 1.0 U/ml, 0.75 U/
mg, respectively.
2.2.2. Three-phase partitioning of watermelon a-galactosidase
The crude watermelon a-galactosidase extract (2 ml containing
0.373 U and 0.5 mg protein) was saturated with 50% (w/v) ammo-
nium sulfate at 25 °C and then vortexed gently to dissolve the salt.
The pH of the system was adjusted to pH 6.0 by the addition of
conc. HCl. After the addition of t-butanol with the ratio of 1:1 (v/
v), the mixture was vortexed gently for 1 min and then allowed
to stand for 1 h at 25 °C. Afterwards, the mixture was centrifuged
at 4000 rpm for 10 min at 4 °C and the three phases (upper organic
phase, middle interfacial precipitate and lower aqueous phase)
were observed. The t-butanol layer (upper phase) was removed
carefully with a pasteur pipette. The aqueous layer (bottom phase)
was collected by piercing the interfacial precipitate layer (middle
phase) using a pipette. The interfacial precipitate containing
a-galactosidase was collected and then dissolved in 1 ml of sodium
citrate buffer (0.05 M, pH 6.0). The lower aqueous layer was also
collected, dialyzed against same buffer and then analyzed for
a-galactosidase activity and total protein content.
Some parameters affecting to the TPP of a-galactosidase were
also searched. The effect of salt (MgSO4, (NH4)2SO4, NaCl, Na2HPO4,
Na2SO4, K2HPO4) and organic solvent (n-butanol, dioxane, n-propa-
nol, t-butanol, isopropanol) type to the partitioning of a-galactosi-
dase were investigated. Beside of this, percent saturations of
ammonium sulfate (20%, 30%, 40%, 50% and 60%, w/v), crude en-
zyme extract to t-butanol ratios (1:0.5, 1:1, 1:1.5 and 1:2, v/v)
and pH of the medium (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0) on
the partition behavior of a-galactosidase were also searched. The
optimized best conditions which resulted into maximum recovery
were used as standard purification procedure for a-galactosidase.
The activity of the crude extract initially added (0.373 U) was taken
as 100%. The blank system was prepared containing ammonium
sulfate, distilled water and t-butanol in the similar manner as for
the crude extract. All the experiments were run in duplicate and
the difference in the readings in triplicates was less than ±5%.
2.2.3. Assay of a-galactosidase activity
a-Galactosidase activity was determined using p-nitrophenyl-
a-D-galactopyranoside (PNPG) as substrate. Reaction mixture con-
sisting of 0.25 ml of 2 mM PNPG, 0.5 ml of sodium citrate buffer
(pH 6.0) and 0.25 ml of enzyme solution was incubated at 37 °C
for 30 min. The reaction was stopped by the addition of 0.2 M so-
dium borate buffer (pH 9.8). The quantity of liberated p-nitrophe-
nol was measured at 400 nm [33].
One unit (1 U) of enzyme activity was defined as the amount of
enzyme which released 1 lmol of p-nitrophenol from PNPG per
minute at pH 6.0 and 37 °C. The data presented for all a-galactosi-
dase activity determinations are mean values of triplicate assay in
which the standard deviations were always smaller than 10%.
2.2.4. Protein determination
Protein concentrations were estimated according to the Coo-
massie Blue G-250 dye binding assay using bovine serum albumin
as the standard protein [34]. Specific activity was expressed as
units per milligram of protein with PNPG as substrate.
2.2.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE)
SDS–PAGE of the protein samples with 12% gel was performed
according to the method of Laemmli [35] with slight modification
using Biorad Mini Protean II electrophoresis unit. For protein stain-
ing, the gel was stained with Coomassie Brilliant Blue R-250 for 1 h
and then destained with 40% (v/v) methanol and 10% (v/v) acetic
acid for 2–3 h. Sigma molecular weight marker mix consists of bo-
vine serum albumin (66 kDa), egg albumin (45 kDa), rabbit muscle
glyceraldehyde 3-P-dehydrogenase (36 kDa), bovine carbonic
anhydrase (29 kDa), bovine pancreatic trypsinogen (24 kDa), soy
bean trypsin (20 kDa) and bovine milk a-lactalbumin (14 kDa)
was used.
2.2.6. Effect of temperature on the activity and stability of partitioned
a-galactosidase
In order to determine the effect of temperature on the activity
of a-galactosidase, activity assays were carried out over the tem-
perature range of 4–80 °C using the standard a-galactosidase assay
at the given temperature. The relative activities (%) were expressed
as the ratio of the a-galactosidase activity obtained at a certain
temperature to the maximum activity obtained at the given tem-
perature range. The termal stability of the enzyme was determined
 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841
by measuring the residual activity of the enzyme exposed to differ-
ent temperatures (4–70 °C) in sodium citrate buffer for 30 min
with continuous shaking. After desired incubation periods enzyme
aliquots were withdrawn and assayed at optimal assay conditions
to determine the residual a-galactosidase activity.
2.2.7. Effect of pH on the activity and stability of partitioned a-
galactosidase
The pH-activity profile of a-galactosidase was studied by incu-
bating samples with PNPG in citrate–phosphate buffer of different
pHs ranging from 2.6 to 7.0, in phosphate buffer of different pHs
ranging from 7.0 to 8.0 and in Tris/HCl buffer of different pHs rang-
ing from 8.0 to 9.0 at 37 °C. In order to determine the pH-stability
of the enzyme, a-galactosidase was incubated in above buffers for
3 h at 4 °C and then the residual activity (%) with respect to control
was assayed under standard activity assay conditions. Each set of
experiment for pH-activity and pH-stability were carried out in
triplicates.
2.2.8. Kinetic constants
The influence of substrate concentration on the a-galactosidase
activity was carried out with the initial concentration of PNPG
ranging from 0.05 to 2.0 mM at pH 6.0 and 37 °C. The maximum
velocity of reaction (Vmax) and Michaelis–Menten constant (Km)
for a-galactosidase were calculated from Lineweaver–Burk plot
which is a plot of 1/V against 1/[S] for systems obeying the Michae-
lis–Menten equation.
3. Results and discusion
Industrial point of a-galactosidases, especially in the food
industry, have important application area. For this reason, a-galac-
tosidases from different sources like; plants, animals and microor-
ganisms were purified using conventional separation and
purification methods (ammonium sulfate fractionation and chro-
matographical steps including gel filtration, ion-exchange, hydro-
phobic interaction and affinity chromatography) [36–40]. Many
of these protocols are multistep, time-consuming, expensive and
difficulty scale enlargement. In the present study, we have used
three-phase partitioning (TPP) for direct one-step purification of
a-galactosidase from watermelon (Citrullus vulgaris) fruit to over-
come mentioned drawbacks of classical purification methods.
TPP is an effective bioseparation technique for the extraction, con-
centration and purification of enzymes [15,17].
3.1. Effect of different salts and organic solvents on partitioning of a-
galactosidase
Distribution behavior of biomolecules in TPP system is a very
complex phenomenon due to many factors which affect the sepa-
ration of proteins in TPP system. Generally, pH, temperature, salt
and organic solvent type and their concentrations can precipitate
proteins at the interphase of TPP system [17]. Therefore, the effect
of various process parameters on partitioning of a-galactosidase
were analyzed to determine the best purification conditions for
TPP. In a TPP system, the type of salt and also its concentration
are an important parameters for effective partitioning of proteins
to the middle phase of the system [15]. In the present study, differ-
ent partitioning experiments with various salts ((NH4)2SO4, MgSO4,
Na2SO4, Na2HPO4, K2HPO4, NaCl) (Table 1) and organic solvents (t-
butanol, n-butanol, n-propanol, isopropanol and dioxane) (Table 2)
for a-galactosidase were performed. TPP systems (50% (w/v) salt
concentration and 1:1 (v/v) t-butanol in the presence) were pre-
pared by using magnesium sulfate as chaotropic salts, sodium sul-
fate, sodium phosphate, potassium phosphate and ammonium
837
Table 1
Effect of different salt types on the degree of purification and activity recovery of
watermelon a-galactosidase.a
Salt type Purification fold Activity recovery (%)
(NH4)2SO4 2.65 75.0
Na2SO4 1.48 71.4
MgSO4 1.08 9.1
K2HPO4 0.79 12.0
NaCl 0.78 15.1
Na2HPO4 0.21 1.3
a
The various type of salts (50%, w/v) were added to the crude extract of water-
melon a-galactosidase (2 ml containing 0.373 U) and then pH was adjusted to
pH 5.5. This was followed by addition of t-butanol in a ratio of 1:1 (v/v) (crude
extract: t-butanol). Three phases formed were collected separately. The upper
phase was removed and then the lower aqueous phase and interfacial precipitate
were tested for enzyme activity and protein amount. Each experiment was carried
out in triplicate and the difference in the readings was less than ±5%.
Table 2
Effect of different organic solvent types on the degree of purification and activity
recovery of watermelon a-galactosidase.a
Organic solvent type Purification fold Activity recovery (%)
n-Butanol 2.48 59.8
Dioxane 1.56 57.6
n-Propanol 3.01 59.3
t-Butanol 2.63 69.2
Isopropanol 2.75 55.8
a
The ammonium sulfate (50%, w/v) was added to the crude extract of water-
melon a-galactosidase (2 ml containing 0.373 U) and then pH was adjusted to pH
5.5. This was followed by addition of organic solvents in a ratio of 1:1 (v/v) (crude
extract: organic solvent). Three phases formed were collected separately. The upper
phase was removed and then the lower aqueous phase and interfacial precipitate
were tested for enzyme activity and protein amount. Each experiment was carried
out in triplicate and the difference in the readings was less than ±5%.
sulfate as a cosmotrophic salt and sodium chloride as a neutral salt
for a-galactosidase partitioning and the effect of salt type to a-
galactosidase distribution was investigated. As is seen from the Ta-
ble 1, an effective partitioning for a-galactosidase was obtained by
using ammonium sulfate as kosmotropic salt with 2.65-fold purifi-
cation and 75% activity recovery. Except from sodium sulfate, the
other salts gave lower fold purification and activity recovery val-
ues. Various salt types were tested in TPP systems, but ammonium
sulfate was often provided the most efficient on protein partition-
ing [16,19, 41]. Dhananjay and Mulimani [18] have also found the
ammonium sulfate is the best for the partitioning of a-galactosi-
dase from Aspergillus oryzae with TPP. As also reported before by
other researchers, in TPP process salting out of a protein by sulfate
is linked to kosmotropy, osmotic stressor, ionic strength effects and
the binding of sulfate to cationic sites of a protein [15].
After determining the type of salt, the new TPP systems (50%
(w/v) ammonium sulfate saturation and 1:1 (v/v) different organic
solvents in the presence) were established to determine the most
effective type of organic solvent for a-galactosidase partitioning.
As shown in Table 2, in comparison to other solvents t-butanol
was more effective organic solvent and had 2.63-fold and 69.2%
recovery for partitioning of a-galactosidase. Isopropanol gave
higher fold (2.75) value, but activity recovery was much lower
(55.8%). Various C4 alcohols can be used as phase-forming organic
solvent in TPP systems. One of these alcohols is t-butanol that
make three-phase layers and remove efficiently small molecular
weight compounds, such as; lipids, phenolics, pigments and en-
zyme inhibitors. It is also a very effective solvent as kosmotropic
and clustering agent at room temperature. Due to its size and
branched structure, t-butanol does not easily permeate inside the
folded protein molecules and hence does not cause denaturation
838 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841
[16,18,42,43]. Similar results have also been obtained with t-buta-
nol for a-galactosidase partitioning [18].
3.2. Three-phase partitioning of a-galactosidase
After the determining the type of salt and organic solvent as
ammonium sulfate and t-butanol, respectively, various process
parameters including percent saturations of ammonium sulfate
(20%, 30%, 40%, 50% and 60%, w/v), crude extract to t-butanol ratios
(1:0.5, 1:1, 1:1.5 and 1:2, v/v) and pHs (3.0, 4.0, 5.0, 5.5, 6.0, 6.5 and
7.0) were analyzed for the partitioning of a-galactosidase. The ef-
fects of ammonium sulfate saturation, crude enzyme extract to t-
butanol ratio and pH of the system on the degree of purification
and on the activity recovery of a-galactosidase are presented in
Figs. 1–3, respectively. The results showed that, all the mentioned
process parameters are very important and affective on the parti-
tioning and purification of watermelon a-galactosidase. The effi-
ciency of the salting out of proteins will first depend on
ammonium sulfate saturation and second on the net charge of
the proteins [15–17].
TPP systems are prepared for a-galactosidase, while ammonium
sulfate saturation of 20% (w/v) and enzyme:t-butanol ratio of 1:1
(v/v) enzyme preferred to stay at the lower aqueous phase. Such
results obtained, a new TPP step is applied by using lower aqueous
phase of first TPP system [28,29]. However, this usually increases
cost and process time. To get rid of the second step of TPP, ammo-
nium sulfate saturation was increased from 20% to 60% (w/v). In
general, in order to obtain the maximum amount of the desired
protein in the interfacial precipitate ammonium sulfate saturation
changes between 20% and 60% (w/v). The salt saturations lower
than 20% (w/v) generally result in poor protein recovery using
TPP. Above 20% (w/v) ammonium sulfate saturation in TPP system,
enzyme is moving from lower phase to upper phase. The effect of
ammonium sulfate saturation was studied by maintaining the ratio
of crude extract to t-butanol ratio constant (1:1) (v/v) and varying
the saturation of ammonium sulfate in the ranges of 20%, 30%, 40%,
50% and 60% (w/v) (Fig. 1). Shown from Fig. 1, the maximum fold
purification of 2.67-fold along with 76.7% recovery of a-galactosi-
dase activity in the interfacial phase was obtained with 50% (w/v)
ammonium sulfate saturation. When the amount of salt and ratio
of enzyme:t-butanol were increased, a high protein amount was
obtained in the interphase and also purification degree and activity
Fig. 1. Effect of varying saturations of ammonium sulfate on the degree of
purification and activity recovery of watermelon a-galactosidase [the crude extract
(2 ml containing 0.373 U) was brought to different levels of saturation w.r.t.
ammonium sulfate (20%, 30%, 40%, 50% and 60%) and t-butanol was added in the
ratio of 1:1 (v/v) with respect to the volumes of the aqueous extract. The lower
aqueous phases and interfacial precipitates were collected separately and
analyzed].
Fig. 2. Optimization of crude extract to t-butanol ratio for the recovery of
watermelon a-galactosidase [various amount of t-butanol was added to crude
extract (2 ml containing 0.373 U and saturated with 50% ammonium sulfate) in the
following volumetric ratios viz. 1:0.5, 1:1, 1:1.5 and 1:2. The lower aqueous phases
and interfacial precipitates were collected separately and analyzed].
recovery were reduced. TPP systems with 20%, 30%, 40% and 60%
(w/v) ammonium sulfate saturations and ratio of enzyme extract
to t-butanol of 1:1 (v/v) gave 0.21-, 0.52-, 2.16- and 2.21-fold puri-
fication with 2.6%, 4.8%, 65.2% and 74% activity recovery of a-galac-
tosidase, respectively (Fig. 1). As is also seen from the figure, when
the ammonium sulfate saturation lower than 50% (w/v) a-galacto-
sidase recovery was very poor in TPP system.
The crude extract:t-butanol ratio which is also very important
in TPP was optimized. In order to determine the best ratio, the
ammonium sulfate saturation was fixed to 50% (v/v) and the crude
extract:t-butanol ratio was varied from 1:0.5 to 1:2 (v/v). The best
a-galactosidase purification fold (2.67) and high activity recovery
(76.7%) were obtained from the interphase of the TPP system hav-
ing the ratio of the crude extract to t-butanol ratio of 1:1 (v/v) at
50% (w/v) ammonium sulfate saturation (Fig. 2). As is also shown
in Fig. 2, with an increase in t-butanol volume the purification fold
and activity recovery at the interphase of the system were de-
creased. In contrast to this, when the amount of t-butanol is less,
it does not adequately synergize with ammonium sulfate [15,19].
Fig. 3. Influence of pH on the degree of purification and activity recovery of
watermelon a-galactosidase [ammonium sulfate (50%, w/v) was added to the crude
extract of a-galactosidase (2 ml containing 0.373 U). The pH of the medium was
adjusted to different pH values. This was followed by addition of t-butanol in a ratio
of 1:1 (crude extract to t-butanol). The lower aqueous phases and interfacial
precipitates were collected separately and analyzed].
 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841 839

Another important parameter when performing TPP system is

pH of the medium and isoelectric point (pI) of target protein. The
basic mechanism of TPP is based on the binding of sulfate anion
to the cationic sites in the proteins. This is significantly influenced
by the pH. Distribution and partitioning of biomolecules in TPP
systems changes with pH due to electrostatic interactions between
phases and charged protein. For this reason, the system pH should
be optimized. Generally, four or five different pHs ranging between
3.0 and 7.0 could be suitable for screening the distribution of pro-
teins in TPP system [15,25,29]. The isoelectric point (pI) of a pro-
tein determine the distribution of the protein in TPP system.
When the pH of the TPP process is lower than the pI of the protein,
the desired protein will be distributed to the interphase [15]. The
effect of pH on the partitioning of a-galactosidase was studied by
maintaining the salt saturation (50%, w/v) and also crude extract
to t-butanol ratio (1:1) (v/v) constant. The enzyme extract was first
saturated with 50% (w/v) ammonium sulfate and then the pH of
the system was adjusted to desired pH. a-Galactosidase was effi-
ciently precipitated at pH 5.5 in the interphase with leaving most
of contaminant proteins in the aqueous phase. TPP behavior of
watermelon a-galactosidase with respect to pH is shown in
Fig. 4. SDS–PAGE analysis of watermelon a-galactosidase (lane 1; molecular
Fig. 3. The recovery of a-galactosidase increased from pH 3.0 to weight markers (14–66 kDa), lane 2; TPP purified a-galactosidase (middle phase),
pH 5.5. At pH 5.5 gave a maximum 2.67-fold and 76.7% activity lane 3; TPP-bottom phase).
recovery of a-galactosidase in the interphase of the system. The
overall purification of a-galactosidase from watermelon by TPP is increasing temperature. But after a certain temperature activity
summarized in Table 3. As understood from the obtained results, is decreased due to denaturation of the enzyme protein
it can be said that a-galactosidase has tendency to concentrate in [28,29,33]. The optimum temperature of watermelon a-galactosi-
the interphase of the TPP system that is releated to its structure. dase was found to be 60 °C (Fig. 5). As is also seen from the figure,
Several a-galactosidases have been purified by TPP with different when the temperature was further increased the activity of the en-
purification folds and yields. Çalcı et al. [29] and S
ß en et al. [28] have zyme was reduced significantly. The enzyme retained more than
reported 80% and 127% activity yields of a-galactosidase corre- 50% of its initial activity at a broad temperature range of 40–
sponding to 4.3- and 6.2-fold purifications, respectively. 65 °C. The results compare well with the previous studies con-
ducted by several authors using different a-galactosidases. For
3.3. Biochemical characterisation of watermelon a-galactosidase example, S ß en et al. [28] found the optimum temperature of a-
galactosidase purified from pepino with TPP as 50 °C. Similarly,
3.3.1. SDS–PAGE analysis maximum activity for tomato a-galactosidase was determined at
SDS–PAGE analysis of the partitioned a-galactosidase showed 37 °C [29]. In general, plant a-galactosidases show high activity
that, the enzyme was nearly homogenous with a molecular weight in the temperature range of 37–60 °C depending to their source
corresponding to 45 kDa (Fig. 4). The molecular weights of a-galac- and also incubation time [19,33,46].
tosidases are varying according to their source and the applied Thermostability of the purified a-galactosidase was carried out
method. Generally, the molecular weights of plant a-galactosi- by incubating the enzyme in the absence of substrate at various
dases are between 30 and 100 kDa [39,44,45]. For examples, the temperatures for 30 min and the results are shown in Fig. 6. As is
molecular weight of a-galactosidase were found as 38 kDa and seen from Fig. 6, the watermelon a-galactosidase is also very stable
34 kDa purified from pepino (Solanum muricatum) [28] and tomato between the temperature range of 4–60 °C. The enzyme retained
(Lycopersicon esculentum) [29] by using TPP, respectively. 57% of its initial activity at 60 °C but relative activity was decreased
sharply at temperatures above 60 °C. This finding is also in agree-
3.3.2. Effect of temperature on the activity and stability of a- ment with our previous studies. In these studies, we have found
galactosidase that pepino a-galactosidase [28] and tomato a-galactosidase [48]
The effect of temperature on a-galactosidase activity was stud- are very stable in the temperature range of 37–55 °C and 4–
ied in the temperature range of 4–80 °C. Enzymes which are large 45 °C, respectively.
and very complex molecules, are protein structure. Their three-
dimensional structures must be protected for the protection of en- 3.3.3. Effect of pH on the activity and stability of a-galactosidase
zyme activity. Temperature is one of the important parameter that One of the other important parameter effecting to the enzyme
affect the activity of enzyme. The reaction rate increases with activity is pH. pH optima of enzymes is affected by many factors,

Table 3
Overall purification of a-galactosidase from watermelon by three-phase partitioning.a

Step Total activity (unit) Total protein (mg) Specific activity (unit/mg) Purification fold Activity yield (%)
Crude extract (85%, w/v, ammonium sulfate fraction) 0.373 0.500 0.75 1.00 100.0
TPP-interfacial precipitate 0.287 0.143 2.00 2.67 76.7
TPP-aqueous phase 0.012 0.084 0.14 0.19 3.2
a
The ammonium sulfate (50%, w/v) was added to the crude extract of watermelon a-galactosidase (2 ml containing 0.373 U) and then pH was adjusted to pH 5.5. This was
followed by addition of t-butanol in a ratio of 1:1 (v/v) (crude extract: t-butanol). Three phases formed were collected separately. The upper phase was removed and then the
lower aqueous phase and interfacial precipitate were tested for enzyme activity and protein amount. Each experiment was carried out in triplicate and the difference in the
readings was less than ±5%.
840 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841
Fig. 5. Effect of temperature on the activity watermelon a-galactosidase (activities
were assayed at indicated temperatures by using PNPG).
Fig. 6. Thermal stability of watermelon a-galactosidase (after 30 min. incubation at
indicated temperatures activities were assayed at 37 °C by using PNPG).
Fig. 7. Effect of pH on the activity of watermelon a-galactosidase (after incubation
at indicated pHs activities were assayed at 37 °C by using PNPG).
such as incubation period, the buffer type and its concentration
and ionic strength. Biochemical reactions taking place in vivo in
an aqueous environment, pH affects the activity of the enzyme
due to the charge status [33]. As shown in the Fig. 7, optimum
pH of watermelon a-galactosidase is determined as pH 6.0. En-
zyme is also very active over a broad pH range of pH 2.0–7.5. a-
Galactosidases have different pH-activity profiles depending on
the source of the enzyme. These findings are in correlation with
previous reports for plant a-galactosidases. Generally, the opti-
mum pH values for plant-derived enzymes are range from pH 3.5
to 6.5 [19,28,29].pH stability of an enzyme is also affected by same
Fig. 8. pH stability of watermelon a-galactosidase (after 3 h incubation at indicated
pHs activities were assayed at 37 °C by using PNPG).
Fig. 9. Lineweaver–Burk plot of watermelon (activities were assayed at 37 °C by
using different concentrations of PNPG).
factors as in the pH optimum (Fig. 8). As shown in the figure, en-
zyme is fairly stable between pH 4.5 to 7.0 and remains initial
activity more over 80%. This result is especially very important
for industrial applications of the enzyme. Generally, a-galactosi-
dases remain fairly stable over a wide pH range. Similar
pH-stability and pH-activity results were reported for various
a-galactosidases [3,19,28,29,33]. For example, enzyme activity is
very stable over the pH range of 4.0–7.0 for A. oryzae and A. niger,
5.0–7.0 for C. cladosporides[3], 4.0–7.0 for pepino [28], 2.5–5.5 for
tomato [29] a-galactosidases.
3.3.4. Determination of kinetic constants
The enzyme kinetics obeyed simple Michaelis–Menten kinetics.
Michaelis–Menten constant (Km) and maximum velocity of reac-
tion (Vmax) for PNPG was determined using the described condi-
tions of the a-galactosidase assay and a PNPG concentration in
the range from 0.05 to 2.0 mM. Km and Vmax were calculated from
Lineweaver–Burk graph as 0.14 mM and 0.12 U, respectively
(Fig. 9). Similar results have been reported by other researchers.
The Km value of a-galactosidase is in agreement with those pre-
sented in literatures [3,19,33,47].
4. Conclusion
Under optimized conditions, a-galactosidase was concentrated
and purified from watermelon (Citrullus vulgaris) with nearly 2.7
purification fold and 76.7% activity recovery by TPP. Ammonium
sulfate and t-butanol were determined as the best suitable compe-
nents of the TPP system. Optimum temperature and pH values of
the enzyme were found as 60 °C and pH 5.5, respectively. Enzyme
 H. Bayraktar, S. Önal / Separation and Purification Technology 118 (2013) 835–841
had high activity recovery at a range of 40–65 °C and pH 4.5–7.0.
Km and Vmax of a-galactosidase were calculated as 0.14 mM and
0.12 U. As a result of these, watermelon is a good source for a-
galactosidase. With necessary optimization, TPP is also possible
and useful for purification of a-galactosidases. Biochemical proper-
ties of the enzyme have showed that, enzyme should be found a
potential for various industrial applications.
Acknowledgement
This work has been funded by the Ege University Research
Foundation under Project 2010 FEN 006. The authors would like
to express their sincere thanks to Ege University, Turkey for finan-
cial support.
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